Mechanisms and Microenvironment Investigation of Cellularized High Density Gradient Collagen Matrices via Densification

Biological tissues and biomaterials are often defined by unique spatial gradients in physical properties that impart specialized function over hierarchical scales. The structure of these materials forms continuous transitional gradients and discrete local microenvironments between adjacent (or within) tissues, and across matrix–cell boundaries, which is difficult to replicate with common scaffold systems. Here, the matrix densification of collagen leading to gradients in density, mechanical properties, and fibril morphology is studied. High‐density regions form via a fluid pore pressure and flow‐driven mechanism, with increased relative fibril density (10×), mechanical properties (20×, to 94.40 ± 18.74 kPa), and maximum fibril thickness (1.9×, to >1 μm) compared to low‐density regions, while maintaining porosity and fluid/mass transport to support viability of encapsulated cells. Similar to the organization of the articular cartilage zonal structure, it is found that high‐density collagen regions induce cell and nuclear alignment of primary chondrocytes. Chondrocyte gene expression is maintained in collagen matrices, and no phenotypic changes are observed as a result of densification. Collagen densification provides a tunable platform for the creation of gradient systems to study complex cell–matrix interactions. These methods are easily generalized to compression and boundary condition modalities useful to mimic a broad range of tissues.     

Layer‐by‐Layer Nanoparticles as an Efficient siRNA Delivery Vehicle for SPARC Silencing

Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC‐siRNA in the bilayers of layer‐by‐layer (LbL) nanoparticles (NPs) with poly(L‐arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO‐siRNA and SPARC‐siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC‐gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT‐PCR. The multilayer SPARC‐siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC‐gene knock down was achieved by using 0.4 pmole siRNA/μg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real‐time cell proliferation and MTT cell assay, indicated that the SPARC‐siRNA‐loaded LbL NPs are non‐toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non‐viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics.     

Comparison of some characteristics of aerobic granules and sludge flocs from sequencing batch reactors.

Physical, chemical and biological characteristics were investigated for aerobic granules and sludge flocs from three laboratory-scale sequencing batch reactors (SBRs). One reactor was operated as normal SBR (N-SBR) and two reactors were operated as granular SBRs (G-SBR1 and G-SBR2). G-SBR1 was inoculated with activated sludge and G-SBR2 with granules from the municipal wastewater plant in Garching (Germany). The following major parameters and functions were measured and compared between the three reactors: morphology, settling velocity, specific gravity (SG), sludge volume index (SVI), specific oxygen uptake rate (SOUR), distribution of the volume fraction of extracellular polymeric substances (EPS) and bacteria, organic carbon and nitrogen removal. Compared with sludge flocs, granular sludge had excellent settling properties, good solid-liquid separation, high biomass concentration, simultaneous nitrification and denitrification. Aerobic granular sludge does not have a higher microbial activity and there are some problems including higher effluent suspended solids, lower ratio of VSS/SS and no nitrification at the beginning of cultivation. Measurement with CLSM and additional image analysis showed that EPS glycoconjugates build one main fraction inside the granules. The aerobic granules from G-SBR1 prove to be heavier, smaller and have a higher microbial activity compared with G-SBR2. Furthermore, the granules were more compact, with lower SVI and less filamentous bacteria.     

Growth, structure and oxygen penetration in particle supported autotrophic biofilms.

Particle supported autotrophic biofilms were cultivated in external-loop airlift reactors at two different pumice concentrations. Oxygen microelectrodes were used to investigate substrate transport and conversion. A special flow cell was designed for the measurement of oxygen concentration profiles in the particle supported biofilms under defined hydrodynamic conditions. The oxygen concentration profiles inside the biofilms were found to be steeper at high flow velocities in the bulk phase of the flow cell compared to those at low flow velocities. Furthermore, the oxygen flux increased and the thickness of the concentration boundary layer decreased with increasing flow velocity. This dependence was found to be more pronounced in less dense biofilms out of airlift reactors with lower pumice concentrations. In addition confocal laser scanning microscopy (CLSM) was used to visualize the biofilm structure. The volume fractions of bacteria and extracellular polymeric substances (lectin-specific EPS-glycoconjugates) were measured in living fully hydrated biofilms. Both the microelectrode and CLSM measurement showed the influence of shear stress on particle supported biofilms. A higher particle concentration led to dense biofilms with a homogeneous surface, lower thickness of the concentration boundary layer and steeper oxygen concentration profiles. The combination of both techniques allows a detailed and quantitative characterisation of particle associated biofilm structure and function.     

The root surface as the definitive detail for microbial transformation processes in constructed wetlands--a biofilm characteristic.

It was the goal of the investigations to characterise the biofilm on the plant roots because of the demonstrable major role of these associated bacteria. The essential criteria for the research were to look at the structure of the microbial colonisation (pattern, density) and to determine properties of the rhizoplane biofilm such as thickness and structure. The root material from a hydroponic system, planted with Glyceria maxima and used for nitrogen removal, has been used for the investigations. Several properties of the bacteria became visible due to the application of specific dyes. The evaluation of the samples was performed by scanning confocal laser microscopy (CLSM). It was shown that the microbial colonisation of the root surface of Glyceria maxima was on an unexpected high level and seems to be related mainly to the permeability and therefore to the age of the plant roots. The thickness of the rhizoplane biofilm is remarkably thin; no inactive layers could be observed in contrast to biofilm growing on technical carrier material. Caused by the untypically two-sided supply with nutrients the whole biofilm is in interaction with the surroundings. This indicates the importance of the plant roots for the microbial transformation processes in wetlands and underlines the especialness of the root as carrier for microorganisms.     

Integrated operation of diagnostic and control systems

In fusion research the ability to generate and sustain high performance fusion plasmas gains more and more importance. Optimal combinations of magnetic shape, temperature and density profiles as well as the confinement time are identified as advanced regimes. Safe operation in such regimes will be crucial for the success of ITER and later fusion reactors. The operational space, on the other hand, is characterized by nonlinear dependencies between plasma parameters. Various MHD limits must be avoided in order to minimize the risk of a disruption.Sophisticated feedback control schemes help to tackle this challenge. But these in turn require detailed information on plasma state in time to allow proper reaction. Control system and diagnostic systems therefore must establish a symbiotic relationship to carry out such schemes. Today, all major fusion devices implement such a concept.An implementation of such a concept with sustained integration is presented using the example of ASDEX Upgrade. It covers data communication via a real-time network, synchronization mechanisms for data-driven algorithm execution as well as operational aspects and exception handling for failure detection and recovery. A modular distributed software framework offers standardized user algorithm interfaces, automated workflow procedures and the application of various computer and network hardware components. Designed with a special focus on reliability, robustness and flexibility, it is a sound base for exploring ITER-relevant plasma regimes and control strategies.     

Fretting damage in thin sheets: Analysis of an experimental configuration

A method for evaluating fretting damage in thin sheets was developed for AISI 301 stainless steel in full hard condition in contact with AISI 52100 steel and cast ANSI A356 aluminum. Samples were subjected to fretting and then were subsequently fatigue tested to determine the impact of the fretting damage on fatigue life. A finite element model of the experimental configuration was used to determine the response for the experimental conditions imposed. The values of Fatemi-Socie critical-plane fatigue damage parameter are shown to correspond to the trends in the observed residual fatigue life for contact with AISI 52100 steel.